Serine proteinase inhibitors

ABSTRACT

A serine protease inhibitor, characterized by having a domain with four cysteines, and a sequence of 0 to 20 amino acids is present between the first and second cysteines, or the serine protease inhibitor has a domain with six cysteines, and a sequence of 7 to 20 amino acids is present between the first and second cysteines.

The present invention relates to serine protease inhibitors, cDNA coding for serine protease inhibitors, medicaments containing such inhibitors or their coding nucleic acid, use of the compounds according to the invention for the preparation of medicaments for the treatment of various indications, antibodies or antibody fragments against epitopes of the compounds according to the invention, poly- or oligonucleotides which will hybridize to genes of the compounds according to the invention, a diagnostic agent for detecting the compounds according to the invention, and medicaments containing antibodies or poly- or oligonucleotides according to the invention.

Proteolytic processes play an important physiological role in all organisms; a distinction has to be made between non-specific and specific proteolytic reactions. The former include, for example, the digestion of food in the digestive tract by endopeptidases, and the intracellular degradation of used endogenous substances and phagocytosed materials by lysosomal proteases. Specific proteolyses mostly serve for the conversion of a proenzyme to its active form, as in the conversion of trypsinogen to trypsin, and of chymotrypsinogen to chymotrypsin, and in the callicrein-kinin cascades and the blood clotting cascade. Depending on the structure of the reactive site of the proteinases involved, they are classified into the classes of serine proteases (e.g., chymotrypsin, trypsin, elastase and cathepsin G), aspartate proteases (e.g., cathepsin D, cathepsin E and pepsin), cysteine proteases (e.g., cathepsin B, cathepsin H and cathepsin L), and the metallo-proteases (e.g., collagenase and thermolysin).

In order to be able to correct the proteolytic processes which often proceed in a cascade, the organisms is provided with a number of other proteins, the protease inhibitors (for a survey, see Laskowski and Kato, 1980, and Bode and Huber, 1992). Thus, the liver-synthesized human plasma protease inhibitors α₁-antichymotrypsin and α₁-proteinase inhibitors protect the lung tissue from non-specific attack by the proteinases cathepsin G and elastase from polymorphonu-clear lymphocytes. When the balance between proteases and their specific inhibitors is disturbed, pathological effects may arise. For example, an excess ratio of elastase to α₁-proteinase inhibitor increases the risk of formation of a lung emphysema by a factor of about 20 to 30 in patients with a genetically caused deficiency in this factor as compared to the normal population (Carrel and Owen, 1980). With smokers, the formation of an emphysema is promoted by oxidation of the amino acid methionine which is present in the reactive site of the α₁-proteinase inhibitor by oxidants contained in cigarette smoke (Miller and Kuschner, 1969; Ohisson et al., 1980). Also in the case of infection with Gram-negative bacteria, their endotoxins can cause disintegration of phagocytes and thus the secretion of lysosomal proteases, which may cause an uncontrolled damage to tissues and inflammations due to the increased consumption of protease inhibitors. For this reason, certain protease inhibitors have a high therapeutic potential (see, e.g., Fritz, 1980).

SUMMARY OF THE INVENTION

It has been the object of the present invention to provide further inhibitors of serine proteases. In addition, the genes or cDNA coding for the inhibitors according to the invention should be provided.

A specific feature of the serine protease inhibitors according to the invention is that the serine protease inhibitor has a domain with four cysteines, and a sequence of 0 to 20 amino acids is present between the first and second cysteines, or the serine protease inhibitor has a domain with six cysteines, and a sequence of 7 to 20 amino acids is present between the first and second cysteines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates VAKTI-1 cDNA and its translation product.

FIG. 2 illustrates VAKTI-2 cDNA and its translation product

FIG. 3 graphically illustrates trypsin inhibition using and embodiment of the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, a sequence of 13 amino acids is present between a first and a second cysteine, and/or a sequence of 18 amino acids is present between a second and a third cysteine, and/or a sequence of 2 amino acids is present between a third and a fourth cysteine.

It is particularly preferred that the sequence between a first and a second cysteine be selected from

-   -   HEFQAFMKNGKLF, SEYRKSRKNGRLF,     -   DDFKKGERDGDFI, SEFRDQVRNGTLI,     -   SAFRPFVRNGRLG, SEYRHYVRNGRLP,     -   KEYEKQVRNGRLF, DEFRRLLQNGKLF,     -   SQYQNQAKNGILF, AEYREQMKNGRLS, or     -   NEYRKLVRNGKLA, DEFRSQMKNGKLI         and/or the sequence between a second and a third cysteine be         selected from     -   PQDKKFFQSLDGIMFINK, TRENDPIQGPDGKMHGNT,     -   TRENDPVLGPDGKTHGNK, TREHNPVRGPDGKMHGNK,     -   TRESDPVRGPDGRMHGNK, TRENDPIEGLDGKIHGNT,     -   TRENDPIRGPDGKMHGNL, TRENDPVRGPDGKTHGNK,     -   TRENDPIQGPDGKVHGNT, TRESDPVRDADGKSYNNQ, or     -   TRESDPVRGPDGKTHGNK         and/or the sequence between a third and a fourth cysteine be         selected from     -   AT, AL, AM, SM, or TM.

It is particularly preferred that the serine protease inhibitor according to the invention correspond to one of the following formulas: R₁-C-HEFQAFMKNGKLF-C-PQDKKFFQSLDGIMFINK-C-AT-C-R₂ (SEQ ID NO:30) R₁-C-DDFKKGERDGDFI-C-PDYYEAVCGTDFKTYDNR-C-AL-C-R₂ (SEQ ID NO:31) R₁-C-SAFRPFVRNGRLG-C-TRENDPVLGPDGKTHGNK-C-AM-C-R₂ (SEQ ID NO:32) R₁-C-KEYEKQVRNGRLF-C-TRESDPVRGPDGRMHGNK-C-AL-C-R₂ (SEQ ID NO:33) R₁-C-SQYQNQAKNGILF-C-TRENDPIRGPDGKMHGNL-C-SM-C-R₂ (SEQ ID NO:34) R₁-C-NEYRKLVRNGKLA-C-TRENDPIQGPDGKVHGNT-C-SM-C-R₂ (SEQ ID NO:35) R₁-C-SEYRKSRKNGRLF-C-TRENDPIQGPDGKMHGNT-C-SM-C-R₂ (SEQ ID NO:36) R₁-C-SEFRDQVRNGTLI-C-TREHNPVRGPDGKMHGNK-C-AM-C-R₂ (SEQ ID NO:37) R₁-C-DEFRSQMKNGKLI-C-TRESDPVRGPDGKTHGNK-C-TM-C-R₂, wherein R₁ is NH₂, an amino acid, or a peptide with up to 100 amino acids, and R₂ is COOH, CONH₂, an amino acid, or a peptide with up to 100 amino acids.

It is further preferred that the serine protease inhibitor contains one or more disulfide bridges. It is particularly for it to contain a disulfide bridge between the first and fourth cysteines and/or between the second and third cysteines, or to contain a disulfide bridge between the first and fifth cysteines and/or between the second and fourth cysteines and/or between the third and sixth cysteines.

Preferred representatives of the serine protease inhibitors according to the invention are the compounds HF 6479 and HF 7665, and fragments of proteins VAKTI-1 and VAKTI-2 according to FIGS. 1 and 2.

In addition to the amino acid sequence of the preferred compounds according to the invention, further information about the cDNA coding for the compounds according to the invention can also be seen from FIGS. 1 to 3. In particular, the corresponding motives and primer-hybridizing sites are indicated.

Compound HF 3479 according to the invention has a mass of 6,479 Dalton, and that of HF 7665 is 7,665 Dalton; both have been purified from hemofiltrate.

According to the invention, a cDNA coding for the compounds according to the invention, especially a cDNA having the nucleic acid sequence according to FIGS. 1 to 2, is also claimed.

The compounds according to the invention are useful as medicaments. In this case, they are administered together with pharmaceutically acceptable vehicles.

The medicaments according to the invention containing the protease inhibitors according to the invention are preferably administered in amounts of from 1 to 100 mg/kg of the patient's body weight. As the dosage form, all galenic formulations for peptide active substances may be used. The medicaments containing nucleic acids according to the invention are preferably administered in amounts of from 0.1 to 100 mg/kg of body weight of a corresponding patient. In this case, the galenic dosage forms which may be used are those which are suitable for the administration of nucleic acids without rendering the nucleic acids ineffective by metabolic influences before they have reached their site of action. For example, liposomes in which the nucleic acids are contained can be employed as a galenic dosage form.

The compounds according to the invention can be used, in particular, for the treatment of acute or chronic cervix inflammations, inflammations of Bartholin's gland or other vaginal regions, tonsillitis, pharyngitis and laryngitis, acute or chronic inflammatory processes accompanied by excessive formation of mucus and the resulting acute emergency situations, postoperative bleedings due to hyperfibrinolysis, and for the prophylaxis of lung emphysema formation in deficiencies of α₁-proteinase inhibitor.

The compounds according to the invention can be administered in deficiencies of serine protease inhibitors to correct endogenous defects. The nucleic acids may also be used in gene therapy, either directly or coupled to suitable vehicles. Suitable vectors include, in particular, attenuated adenoviruses into which the corresponding genes have been incorporated.

The polypeptides according to the invention, especially VAKTI-I and VAKTI-II, can serve for the preparation of antibodies or antibody fragments. These are simply prepared by the immunization of appropriate mammals. By per se known operations, the antibodies may also be humanized so that such antibodies can also be employed for therapeutic use. Antibodies or antibody fragments can then by employed for the regulation of diseases in which the protease inhibitors are expressed in a pathological way. Also, antisense nucleic acids complementary to the nucleic acids according to the invention may also be employed in therapeutical use in overexpressions of the protease inhibitor genes.

The compounds according to the invention can be easily prepared by per se known methods of peptide or nucleotide synthesis. Preparation of the compounds by genetic engineering is also possible.

Those skilled in the art will recognize that fragments of the polypeptides according to the invention may also be used provided that they retain the inhibitory properties of the serine protease inhibitors. Those skilled in the art know how to find such fragments. Thus, this may be accomplished, for example, by a selected enzymatic cleavage of the compounds according to the invention. Side-chain modified amino acids may also be employed. N- or C-terminally modified polypeptides may also be used. In particular, phosphorylated, glycosylated, methylated, acetylated or similarly modified polypeptides can be employed provided that they do not substantially affect the activity of the serine protease inhibitors.

Derivatives of the nucleic acids according to the invention which have modified triplet structures in accordance with codon usage may also be used. In addition, nucleic acids according to the invention also include those which are more stable towards degradation by nucleases as compared with the native compounds, for example, the corresponding SODN derivatives usually employed in antisense technology to give the antisense structures a more stable design towards enzymatic attack.

Structures homologous to the polypeptides may also be used. In particular, these include polypeptide structures in which amino acids have been exchanged. Thus, for example, conservative amino acid substitutions in highly conserved regions can be considered as follows: any isoleucine, valine and leucine amino acid can be exchanged for any other of these amino acids, aspartate can be exchanged for glutamate and vice versa, glutamine for asparagine and vice versa, serine for threonine and vice versa. Conservative amino acid substitutions in less highly conserved regions can be as follows: Any of the amino acids isoleucine, valine and leucine for any other of these amino acids, aspartate for glutamate and vice versa, glutamine for asparagine and vice versa, serine for threonine and vice versa, glycine for alanine and vice versa, alanine for valine and vice versa, any of the amino acids leucine, isoleucine or valine for methionine, lysine for arginine and vice versa, either of the amino acids arginine or lysine for either of the amino acids aspartate or glutamate, either of the amino acids arginine or lysine for histidine, glutamine for glutamate and vice versa, and asparagine for aspartate and vice versa.

The mode of action of the peptides according to the invention will be illustrated by the following Example.

EXAMPLE

Measurement of Protease Inhibition by HF 7665

Measuring Composition:

-   84 μl measuring buffer (0.1 M HEPES, pH 7.5; 0.5 M NaCl) -   1 μl trypsin (1 mg/ml in 1 mM HCl, 20 mM CaCl₂) -   5 μL-BABNA (6 mg/ml N-α-benzoyl-L-arginine-p-nitroanilide     hydrochloride) -   10 μl protease inhibitor (10 μM or 75 μg/ml HF 7665 in H₂O)

The reaction was started by adding the chromogenic substrate, and the substrate conversion was followed by a photometer at X=405 nm. After about five minutes, 10 μl of protease inhibitor or the corresponding controls were added and the further course of the absorbance observed.

It could be shown that HF 7665 has an inhibitory effect on trypsin in a final concentration of about 1 μM or 7.5 μg/ml. Control experiments with corresponding amounts of BSA (7.5 μg/ml) and acetonitrile/TFA (0.8% ACN/0.001% TFA) did not show any trypsin inhibition. Further, an inhibitory effect of HF 7665 on chymotrypsin could not be observed in a similar test.

FIG. 3 shows that the substrate conversion is reduced by about 30% due to trypsin inhibition after the addition of HF 7665. 

1. A purified, synthesized, or genetically engineered serine protease inhibitor, characterized by having a domain with four cysteines, and a sequence of 13 amino acids is present between the first and second cysteines, a sequence of 18 amino acids is present between the second and third cysteines, and a sequence of 2 amino acids is present between the third and fourth cysteines.
 2. The serine protease inhibitor according to claim 1, characterized in that the sequence of the domain between the first and second cysteines is selected from HEFQAFMKNGKLF (SEQ ID NO: 7), SEYRKSRKNGRLF (SEQ ID NO: 8), DDFKKGERDGDFI (SEQ ID NO: 9), SEFRDQVRNGTLI (SEQ ID NO: 10), SAFRPFVRNGRLG (SEQ ID NO: 11), SEYRHYVRNGRLP (SEQ ID NO: 12), KEYEKQVRNGRLF (SEQ ID NO: 13), DEFRRLLQNGKLF (SEQ ID NO: 14), SQYQNQAKNGILF (SEQ ID NO: 15), AEYREQMKNGRLS (SEQ ID NO: 16), or NEYRKLVRNGKLA (SEQ ID NO: 17), DEFRSQMKNGKLI (SEQ ID NO: 18).
 3. The serine protease inhibitor according to claim 1, characterized in that the sequence between the second and third cysteines is selected from PQDKKFFQSLDGIMFINK (SEQ ID NO: 19), TRENDPIQGPDGKMHGNT (SEQ ID NO: 20, TRENDPVLGPDGKTHGNK (SEQ ID NO: 21), TREHNPVRGPDGKMHGNK (SEQ ID NO: 22), TRESDPVRGPDGRMHGNK (SEQ ID NO: 23), TRENDPIEGLDGKIHGNT (SEQ ID NO: 24), TRENDPIRGPDGKMHGNL (SEQ ID NO: 25), TRENDPVRGPDGKTHGNK (SEQ ID NO: 26), TRENDPIQGPDGKVHGNT (SEQ ID NO: 27), TRESDPVRDADGKSYNNQ (SEQ ID NO: 28), or TRESDPVRGPDGKTHGNK (SEQ ID NO: 29).
 4. The serine protease inhibitor according to claim 1, characterized in that the sequence between the third and fourth cysteines of the domain is selected from AT, AL, AM, SM, or TM.
 5. The serine protease inhibitor according to claim 1, having one of the following formulas: R₁-C-HEFQAFMKNGKLF-C-PQDKKFFQSLDGIMFINK-C-AT-C-R₂ (SEQ ID NO: 30) R₁-C-DDFKKGERDGDFI-C-PDYYEAVCGTDGKTYDNR-C-AL-C-R₂ (SEQ INO: 31) R₁-C-SAFRPFVRGLG-G-TRENDPVLGPDGKTHGNK-C-AM-C-R₂ (SEQ ID NO: 32) R₁-C-KEYEKQVRNGRLF-C-TRESDPVRGPDGRMHGNK-C-AL-C-R₂ (SEQ ID NO: 33) R₁-C-SQYQNQAKNGILF-C-TRENDPIRGPDGKMHGNL-C-SM-C-R₂ (SEQ ID NO: 34) R₁-C-NEYRKLVRNGKLA-C-TRENDPIQGPDGKVHGNT-C-SM-C-R₂ (SEQ D NO: 35) R₁-C-SEYRKSRKNGRLF-C-TRENDPIQGPDGKMHGNT-C-SM-C-R₂ (SEQ ID NO: 36) R₁-SERDQVRNGTLI-C-TREHNPVRGPDGKMHGNK-C-AM-C-R₂ (SEQ ID NO: 37) R₁-C-SEYRHYVRNGRLP-C-TRENDPIEGLDGKIHGNT-C-SM-C-R₂ (SEQ ID NO: 38) R₁-C-DEFRRLLQNGKLF-C-TRENDPVRGPDGKTHGNK-C-AM-R₂ (SEQ ID NO: 39) R₁-C-AEYREQMKNGRLS-C-TRESDPVRDADGKSYNNQ-C-TM-C-R₂ (SEQ NO: 40) R₁-C-DERSQMGKLI-C-TRESDPVRGPDGKTHGNK-C-TM-C-R₂ (SEQ ID NO: 41), wherein R₁ is NH₂, an amino acid, or a peptide with up to 1000 amino acids, and R₂ is COOH, CONH₂, an amino acid, or a peptide with up to 1000 amino acids.
 6. The serine protease inhibitor according to claim 1, characterized by containing a disulfide bridge between the first and fourth cysteines and/or between the second and third cysteines; or a disulfide bridge between the first and a fifth cysteine and/or between the second and fourth cysteines and/or between the third and a sixth cysteine.
 7. The serine protease inhibitor according to claim 1, characterized by being a fragment of VAKTI-1 (SEQ. ID. NO. 1) or VAKTI-2 (SEQ. ID. NO. 2).
 8. A purified, synthesized or genetically engineered serine protease inhibitor, characterized by being HF 6479 (SEQ. ID. NO. 3) or HF 7665 (SEQ. ID. NO. 4).
 9. A medicament containing the serine protease inhibitor according to claim 8 together with a pharmaceutical vehicle.
 10. The medicament according to claim 9, containing from 0.01 to 1000 mg per kg of body weight of the serine protease inhibitor.
 11. Method of using the medicament according to claim 9, wherein the medicament is the serine protease inhibitor, for the treatment of acute or chronic cervix inflammations, inflammations of Bartholin's glands and other vaginal regions, tonsillitis, pharyngitis and laryngitis, acute or chronic inflammatory processes accompanied by excessive formation of mucus and the resulting acute emergency situations, postoperative bleeding due to hyperfibrinolysis, and for the prophylaxis of lung emphysema formation in deficiencies of α₁-proteinase inhibitor. 